J Wood Sci (2002) 48:536-541 The Japan Wood Research Society 2002

Shiro Suzuki Norikazu Sakakibara Toshiaki Umezawa Mikio Shimada Survey and enzymatic formation of of Anthriscus sylvestris

Received: July 25, 2001 / Accepted: December 22, 2001

Abstract Gas chromatography - mass spectrometry ana- cal properties I-3 and various biological activities (e.g., anti- lysis of the fl-glucosidase-treated MeOH extracts of tumor, antimitotic, antiviral2-S). Moreover, biosynthesis of Anthriscus sylvestris showed, based on comparison of the some lignans is closely related to heartwood formation, mass spectra and retention times with those of authentic which is a metabolic event specific to woody plants but not samples, the presence of lignans, yatein, secoisolaricire- to herbaceous plantsY sinol, , , hinokinin, and pluviato- An aryltetralin , , isolated from lide. The existence of small amounts of bursehernin was PodophyIlum plants ~-8 is well known to have antitumor ac- suggested by mass chromatography. In addition, nemerosin tivity. The lignan isolated from Podophyllum hexandrurn is and deoxypodophyllotoxin were tentatively identified by exploited commercially as a source of the semisynthetic comparing the mass spectra with those reported in the lit- anticancer drugs etoposide and teniposide. 2-8'1~ However, erature. Enzyme preparations from A. sylvestr& catalyzed owing to the limited supply of the plant, much attention the formation of and lariciresinol from has been focused on the availability and biosynthesis of coniferyl alcohol. Furthermore, the enzyme preparation the lignan, a Several woody plants (e.g., Juniperus savina, catalyzed the formation of lariciresinol from (2)_ Thujopsis dolabrata, Callitris drurnmondii, Hernandia pinoresinols and the formation of secoisolariciresinol from ovigera) have been known to produce podophyllotoxin or (2)-lariciresinols. Thus, /lariciresinol reductase its congeners. 7 Thus, biotechnological production of the (PLR) activity was detected. Chiral high-performance antitumor lignan by the woody plants is a challenging sub- liquid chromatography analysis showed selective formation ject in the field of wood chemistry and biochemistry. As of (+)-lariciresinol and (-)-secoisolariciresinol from (2)_ the first step, it is critically important to understand the pinoresinols with the A. sylvestris PLR preparation, indicat- biosynthetic mechanisms. However, much remains un- ing that the stereochemical property of A. sylvestris known about the biosynthesis of podophyllotoxin. PLR-catalyzed reduction was similar to those of Forsythia In addition to these woody plants and Podophyllum spp., PLR and Arctium lappa ripening seed PLR. some herbaceous plants such as Linum spp. (especially those belonging to the section Syllinum, including Linum Key words Lignan - Anthriscus sylvestris Biosynthesis flavum and Linum album) 5m'12 and Anthriscus sylvestris 13 19 have been known to produce significant amounts of podophyllotoxin congeners. Studies of lignan biosynthesis in Linum spp., 2~ which produce principally 5- Introduction methoxypodophyllotoxin in addition to podophyllotoxin, 2~ have been reported. Although isolation of podophyllotoxin Biosynthesis of lignans has been studied from many aspects from A. sylvestris has not yet been reported, angeloyl because this class of compounds has peculiar stereochemi- podophyllotoxin has been isolated from the species. 22 In addition, this species produces not only the precursor lignans of podophyllotoxin, deoxypodophyllotoxin (= S. Suzuki N. Sakakibara - T. Umezawa (~) - M. Shimada desoxypodophyllotoxin, anthricin) 23 and yatein, 24 but also Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, the typical heartwood lignans yatein and hinokinin, t7 which Japan Tel. +81-774-38-3628; Fax +81-774-38-3682 are found specifically in the heartwood region in the coni- e-mail: [email protected]~u.ac.jp fers Libocedrus yateensis 25 and Charnaecyparis obtusa, 9 respectively. Thus, knowledge of the lignan biosynthetic Part of this report was presented at the 43rd Lignin Symposium,Fuchu, mechanism in A. sylvestris can be applied to biotechnolo- October 1998 gical production of podophyllotoxin in woody and herba- 537 OH H3CO.~OH H3C O~.,~-.r.-- ~ min by a linear solvent gradient protocol of CH3CN-H20 at .o 2o. t = 0 (23:77) to 15 min, then to 50:50 at t = 20min, and held at this ratio for an additional 5 min. (2) Solvent system B was used for gradient elution at i ml/min by a linear solvent o o. ~" OCH3 OH gradient protocol of CH3CN-H20 at t = 0-6 min from 15:85 Lariciresinol Secoiso- Matairesinol to 17:83; at t = 6-16rain from t7:83 to 20:80; 20:80 at t = L\ I~ lariciresinol 16-20min; at t = 20-26 from 20:80 to 50 : 50; and held at this "~ "OCH3 ratio for an additional 5min. Chiral HPLC separation of OH lariciresinol and secoisolariciresinol was conducted exactly O~ as previously described) ~ Silica gel thin-layer chromatogra- 2Jo L,;o phy (TLC) and silica gel column chromatography employed CH30 Kieselgel 60 F254 (Merck) and Kieselgel 60 (Merck), respec- ~0 tively. All solvents and reagents used were of reagent or . .3 "g5~ 0 HPLC grade unless otherwise mentioned. OR2 H3CO~ q RI=OCH3, R2=CH3,Yatein Hinokinin Nemerosin 0 RI=H, R2=H, Pluviatolide Compounds RI=H, R2=CH3, Bursehernin [9,9-2H2, OC2H3]Coniferyl alcohol,27 (+)-[9,9,9',9'- OH 2H4]pinoresinols,2S (-+)-pinoresinols, 27 (+)-[9,9,9',9'- 2H4]lariciresinols,28 (2_-)-lariciresinols, > (_+)-[9,9,9',9'-aH4] secoisolariciresinols,28 (--)-secoisolariciresinols,27 (-)- matairesinols, 3~ (+)-hinokinins, 9 (+)-pluviatolides, 9 and ( + )-bursehernins 28 were prepared previously. ( _+)-Yateins Oeoxypodo- OCH3 were prepared from (+)-4-demethylyatein benzyl ethers (a phyllotoxin Podophyllotoxin gift of Dr. Shingo Kawai) by debenzylation (10% Pd/C, H2) followed by methylation (diazomethane). Fig. 1. Chemical structures of lignans (_+)-Yateins, ~H-NMR (CDC13): 2.43-2.63 (3H, m, C7:8,s,H), 2.88 (1H, dd, J = 13.8 and 6.3, C7H), 2.92 (1H, dd, J = 13.8 and 5.4, CTH), 3.817 (3H, s, OCH3), 3.819 (6H, s, ceous plants and to studies of heartwood lignan biosynthetic OCH 3 • 2), 3.87 (1H, dd, J = 9.0 and 7.6, C9H), 4.17 (1H, mechanisms. In addition, this species exhibits good growth dd, J = 9.1 and 7.2, CgH), 5.92-5.93 (2H, m, -OCH20-), 6.35 behavior and can be maintained easily in laboratories. (2H, s, C2,6H), 6.45-6.47 (2H, m, C2,,6,H), 6.69 (1U, dd, J = Altogether, A. sylvestris is probably a good plant mate- 0.8 and 7.6, Cs,H). rial for lignan biosynthesis studies to access mechanisms involved in the formation of antitumor and heartwood lignans. As the first step, it is necessary to characterize the Plant materials precursor lignans of yatein and deoxypodophyllotoxin in A. sylvestris. We report here the preliminary survey of lignans Anthriscus sylvestris (L.) Hoffm. (Umbelliferae) plants in this species and the enzyme activities catalyzing forma- were grown from seeds collected in a suburb of Brussels, tion of lariciresinol and secoisolariciresinol, which are up- Belgium in 1996 or at Kyoto University Forest in Ashiu, stream lignans in the biosynthetic pathway (Fig. 1). Kyoto in 1997. The plants were maintained in the experi- mental garden of the Wood Research Institute, Kyoto Uni- versity for about 6 months.

Materials and methods Lignans in Anthriscus sylvestris Instrumentation Aerial parts (including leaves, petioles, racemes) and roots ~H-nuclear magnetic resonance (NMR) spectra were ob- of A. sylvestris were individually harvested and washed with tained with a JNM-LA400MK FT NMR system (JEOL) tap and distilled water. Each sample (0.7-1.6 g) was frozen using tetramethylsilane as an internal standard. Chemical (liquid N2), freeze-dried, disintegrated with scissors, and shifts and coupling constants (J) are given in d and Hertz, then extracted with hot MeOH (11 ml). An aliquot (5 mg) of respectively. Gas chromatography-mass spectrometry (GC- the MeOH extract was treated with fi-glucosidase (from MS) was conducted as previously described. 26 Reversed- almonds; Sigma G-0395) (10 units in 0.5 ml of a 0.1 M NaOAc phase high-performance liquid chromatography (HPLC) buffer at pH 5.0) for 24h at 37~ The reaction mixture was was conducted as previously described 27 with the following extracted with CH2C12 (0.5 ml • 2), and the combined solu- elution conditions: The column was a Waters Novapak C1s tion was evaporated off. The resulting residue was subjected (150 • 3.9mm), which was eluted with two solvent systems: to GC-MS analysis after derivatization with 7,al of N,O- (1) Solvent system A was used for gradient elution at i ml/ bis(trimethylsilyl) acetamide (BSA) for 45 rain at 60~ 538 In a separate experiment, aerial parts of A. sylvestris of the BSA solution was subjected to GC-MS analysis, and (from University Forest in Ashiu) were harvested and the lignans formed were identified and quantified (Table 1). freeze-dried as above. The dried materials (48.7g) were ground with a Waring blender and extracted with hot Enzymatic conversion of (• MeOH (100ml • 10); then the solvent was evaporated off. and (• The MeOH extract (12.3 g) was dispersed in Et20 (50ml • 10), and the Et20-soluble materials were removed. The The reaction mixture consisted of 150/~l of 50mM of Et20-insoluble residue (10.3 g) was suspended in 180ml of (+)-[9,9,9',9'-2H4]pinoresinols in MeOH, lml of 50mM 0.1M NaOAc buffer (pH 5.0) containing 2544 units of/3- NADPH in a 0.1M potassium phosphate buffer (pH 7.0), glucosidase, and the solution was incubated for 24h at 37~ and 16mi of the ceil-free extracts from A. sylvestris leaves. Then the solution was extracted with EtOAc (80ml • 4), The reaction was initiated by adding (+)-[9,9,9',9'-2H4] and the solvent was evaporated off. The EtOAc extract pinoresinols. After incubating for 1 h at 30~ the reaction (721mg) was fractionated into six fractions by silica gel mixture was extracted with EtOAc containing unlabeled column (q~ 30 • 80ram) chromatography. Each fraction was (+)-lariciresinol and (• as internal subjected to GC-MS analysis after trimethylsilyl (TMS) standards. An aliquot of the EtOAc extract was sub- derivatization. jected to GC-MS analysis, and [2H4]lariciresinol and [2Hg]secoisolariciresinol were identified. The remainder was Enzyme preparation submitted to silica gel TLC [solvent, MeOH-CHzC12 (3 : 97)] followed by reversed-phase HPLC (solvent system A for An ammonium sulfate precipitate (0%-70% saturation) the lariciresinol purification, and solvent system B for the was prepared from cell-free extracts of young leaves (in- secoisolariciresinol purification) to give [2H4]lariciresinol cluding petioles) of (from seeds harvested in A. sylvestris and [2H4]secoisolariciresinol. Enantiomeric compositions Belgium) exactly as previously described 3~ and used as an of the deuterium-labeled lignans were determined as enzyme preparation after desalting with a Sephadex G-25 previously described using the corresponding unlabeled (Pharmacia) column preequilibrated and eluted with a (• and (_+)-secoisolariciresinols as internal 0.1M potassium phosphate buffer (pH 7.0). The protein standards, respectively. 264s content of the enzyme preparation was 0.55-8.65mg/ml, In a separate experiment, the results shown in Table 2 being measured by the method of Bradford 32 using bovine were obtained under assay conditions similar to those de- serum albumin as a standard. scribed above but with the following scaled-down constitu- ents: 5#1 of 25mM of (• in Enzymatic conversion of [9,9-2H2, OC2H3]coniferyl alcohol

The reaction mixture was composed of 50#1 of 25 mM [9,9- Table 2. Enzymatic formation of [2H4]lariciresinol from (_+)-[9,9,9',9'- 2H> OC2H3]coniferyl alcohol in a 0.1M potassium phos- 2H4]pinoresinols phate buffer (pH 7.0), 50~1 of 50mM NADPH in the same Cofactor [2Ha]Lariciresinol formation buffer, 25#1 of 7.6mM H202 in the same buffer, and 120#1 (nmolh lmg-1 protein) of the cell-free extracts from A. syIvestris. The reaction was initiated by adding H202. After incubation for lh at 30~ Complete assay NADPH 6.18 the reaction mixture was extracted with EtOAc containing Control assay~ unlabeled (--)-pinoresinol, (• and (• None 0 secoisolariciresinol as internal standards. The EtOAc ex- Denatured enzyme/NADPH 0 tract was dried under high vacuum and dissolved in 7#1 of Control assay refers to a complete assay but with omission of either a BSA. After standing at 60~ for 45min, an aliquot (0.8#1) cofactor or the denatured enzyme (boiled for 10min)

Table 1. Enzymatic formation of [2H10]pinoresinol, [2H10]lariciresinol, and [2H10]secoiso- lariciresinol from [9,95H> OC2H3]coniferylalcohol Cofactor [2Hzo]Pinoresinol [2Hlo]Lariciresinol [2Hio]Secoisolarici- formation b formation b resinol formationb Complete assay H202/NADPH 20.7 13.6 36.0 Control assay~ H202 144.0 0 0 NADPH 11.1 10.7 16.9 Denatured enzyme/ 8.2 0 0 HzO2/NADPH Control assay refers to a complete assay but with omission of either cofactors or the denatured enzyme (boiled for 10 rain) bExpressed in nanomoles per hour per milligram of protein 539 MeOH, 50#1 of 50mM NADPH in a 0.1 M potassium phos- phate buffer (pH 7.0), and 200M of the enzyme preparation. Pino- DyO-y~-~OCD3 O~.-Oy ~'L~OCD3 (+-)-[9,9,9',9'-2H4]Lariciresinols were incubated under resinol-dl0 D ~ O _ D the same assay conditions as above, except that 5#l of 25raM (+)-[9,9,9',9'-2H4]lariciresinols were used as "202 ,0" T.... HO~^_ Lar!ci- substrates. UUU 3 UUL/3 resinol-dlo NADPH "~ "OCD3 D D OH Results and discussion DaCO'~~ OH Coniferyl + H O"~'Y~:'g H alcohol-ds The GC-MS analysis of the fi-glucosidase-treated MeOH Secoiso- '<.~-N-- extracts of the aerial parts and roots of A. sylvestris showed lar,clresmol-dlo.... OHT UUU3 the presence of the lignans yatein and secoisolariciresinol, which were identified by comparing of the mass spectra and Fig. 2. Enzymatic conversion of [9,9-2H2, OC2H:~]coniferylalcohol to retention times on GC with those of authentic samples. [2H10]pinoresinol, [2H10]lariciresinol,and [~H~0]secoisolariciresinol Yatein: MS m/z (%): 400([M] § 100), 368(20), 239(10), 223(10), 181(88), 135(31) Table 3. Enzymatic formation of [2H4]secoisolariciresinol from (_+)- Secoisolariciresinol (TMS ether): MS m/z (%): 650([M] +, [9,9;9',9'-2H4]lariciresinols 20), 368(43), 560(10), 470(22), 261(52), 209(100), 179(21) Cofactor [2H4]Secoisolariciresinol formation In addition, two GC peaks, A and B, were detected. (nmolh < nag i protein)

A: MS, m/z (%): 398([M] +, 23), 263(100), 235(4), 207(18), Complete assay 176(12), 161(5), 135(55), 209(100), 179(21) NADPH 47,3 B: MS, m/z (%): 398([M] +, 100), 283(5), 230(13), 185(18), Control assay~ None 3.2 181(30), 173(21) Denatured enzyme/NADPH 11.8 The mass spectra of peaks A and B essentially matched data a Control assay refers to a complete assay but with omission of either a in the literature for the mass spectra of nemerosin 16 and cofactor or the denatured enzyme (boiled for 10 min) deoxypodophyllotoxin,33 respectively, which were previ- ously isolated from Anthriscus spp. t3q9 When partially fractionated MeOH extracts after by GC with those of unlabeled authentic samples, as pre- glucosidase treatment from aerial parts of A. sylvestris by viously reported. 26'27'34 Dehydrogenated dimers other than silica gel column chromatography were analyzed by GC- pinoresinol were not investigated. Next, we incubated (_+)- MS, the lignans lariciresinol, matairesinol, hinokinin, and [9,9,9',9'-2H4]pinoresinols with the enzyme preparation in pluviatolide were identified by comparing the mass spectra the presence of NADPH because pinoresinol/lariciresinol and retention times with those of authentic samples. reductase (PLR) has been known to be involved in the production of lariciresinol and secoisolariciresinol from Lariciresin01 (TMS ether): m/z (%): 576([M] +, 41), 561 (10), pinoresinol. 3~ GC-MS analysis indicated that 486(26), 324(31), 277(31), 223(46), 209(45), 179(19) [2H4]lariciresinol was produced during the incubation Matairesinol (TMS ether): MS m/z (%): 502([M] +, 94), (Table 2). [2H4]Lariciresinol was not formed with the omis- 179(37) 293(4), 209(100), sion of NADPH or with denatured enzyme, demonstrating (%): 354([M] ~, 58), 135(100) Hinokinin: MS m/z that this reaction is enzymatic and requires NADPH as a Pluviatolide (TMS ether): MS m/z (%): 428([M] +, 85), cofactor. [2H4]Secoisolariciresinol was not detected in this 179(28), 135(26) assay, probably due to the low enzyme activity. Therefore, In addition, the presence of small amounts of bursehernin we next incubated (_+)-[9,9,9',9'-2H4]tariciresinols with the was suggested by comparing mass chromatograms with preparation in the presence of NADPH, which afforded those of the synthesized authentic sample. Secoisolarici- [2H4]secoisolariciresinol (Table 3). Control assays resulted resinol, lariciresinol, matairesinol, and pulviatolide were in insignificant specific activity, demonstrating that this identified for the first time in Anthriscus spp. reaction is enzymatic and needs NADPH as a cofactor. Recently, it has been found that there is great stere- These results demonstrate pinoresinol/lariciresinol reduc- ochemical diversity in upstream steps of lignan biosynthesis, tase (PLR) activity in this species. In a separate experiment, and it is important to examine the stereochemical property when we carried out the scaled-up incubation of (-+)- of enzymatic reactions of the upstream steps in A. sylvestris. [9,9,9',9'-2H4]pinoresinols with the enzyme preparation When [9,9-2H2, OC2H3]coniferyl alcohol was incubated in the presence of NADPH, both [2H4]lariciresinol and with the A. sylvestris enzyme preparation in the presence [2H4]secoisolariciresinol were formed, and their enantio- of NADPH and H202, the lignans [2H~0]pinoresinol, meric compositions were determined as follows: [2H10]lariciresinol, and [2H10]secoisolariciresinol were [2H4]lariciresinol, 93% e.e. in favor of (+)-enantiomer; formed (Table 1, Fig. 2). They were identified by comparing [2H4]secoisolariciresinol, 95% e.e. in favor of (-)- their mass spectra as TMS ethers and the retention times enantiomer (Fig. 3). 540 (+-)-Pinoresinols-d4 2. Umezawa T (2001) Biosynthesis of lignans and related phenyl- propanoid compounds (in Japanese). Regul Plant Growth Dev NADPH 1 36:57-67 3. Umezawa T (1997) Lignans. In: Higuchi T (ed) Biochemistry and molecular biology of wood. Springer-Verlag, Berlin, pp 181-194 4. MacRae WD, Towers GHN (1984) Biological activities of lignans. OCH 3 OCH 3 Phytochemistry 23:1207-1220 5. Umezawa T (1996) Biological activity and biosynthesis of lignans (in Japanese). Mokuzai Gakkaishi 42:911-920 f~'~ %'OH > I-/6-OH 6. Ayres DC, Loike JD (1990) Lignans: chemical, biological and clini- HO" "~ HO~ "~ cal properties. Cambridge University Press, Cambridge OCH3 OCH 3 7. Sackett D (1993) Podophyllotoxin, steganacin and combretastatin: natural products that bind at the colchicine site of tubulin. (+)-Lariciresinol-d4 (-)-Lariciresinol-d4 Pharmacol Ther 59:163-223 (93% e.e.) 8. Canel C, Moraes RM, Dayan FE, Ferreira D (2000) Molecules of + interest: podophyllotoxin. Phytochemistry 54:115-120 HDD 9. Takaku N, Choi D-H, Mikame K, Okunishi T, Suzuki S, Ohashi H, H3CO~o H H3CO~-~OHH D D Umezawa T, Shimada M (2001) Lignans of Chamaecyparis obtusa. JI _1 I J Wood Sci 47:476-482 10. Cragg G, Boyd M, Khanna R, Newman D, Sausville E (1999) Natural product drug discovery and development: the United States National Cancer Institute role. In: Romeo J (ed) Recent "OCH3 "~ -OCH3 advances in phytochemistry. Kluwer Academic/Plenum, New OH OH York, pp 1-29 (.)-Secoisolarici- (+)-Secoisolarici- 11. Konuklugil B (1996) Aryltetralin lignans from genus Linum. resinol-d 4 (95% e.e.) resinol-d4 Fitoterapia 67:379-381 12. Broomhead AJ, Dewick PM (1990) Aryltetralin lignans from Fig. 3. Enantiomeric compositions of [2H4]lariciresinol and Linum flavum and Linum capitatum. Phytochemistry 29:3839-3844 [2Hg]secoisolariciresinol formed enzymatically from (+_)-[9,9,9',9'- 13. Noguchi T, Kawanami M (1940) The active components of the 2H4]pinoresinols Umbelliferae. X. Components of Anthriscus sylvestris (in Japa- nese). Yakugaku Zasshi 60:629-636 14. Kozawa M, Morita N, Hata K (1978) Chemical components of the roots of Anthriscus sylvestris Hoffm. I. Structures of an acyloxy- The PLR activity together with enzymatic formation of carboxylic acid and a new phenylpropanoidester, anthriscusin (in the lignans from coniferyl alcohol accorded well with those Japanese). Yakugaku Zasshi 98:1486-1490 with Arctium lappa 26'38 and Forsythia spp. 27'35-39 In addition, 15. Kurihata T, Kikuchi M, Suzuki S, Hisamichi S (1978) Studies of the constituents of Anthriscus sylvestris HOFFM. I. On the compo- the PLR-catalyzed selective formation of (+)-lariciresinol nents of the radix (in Japanese). Yakugaku Zasshi 98:1586-1591 and (-)-secoisolariciresinol from ( +)-pinoresinols with the 16. Turabelidze DG, Mikaya GA, Kemertelidze P, Vul'son NS (1982) A. sylvestris enzyme preparation suggested that the stere- Lignan lactones from the roots of wild chervil Anthriscus nemerosa (Bieb.) Spreng. Soy J Bioorg Chem 8:374-379 ochemical property of A. sylvestris PLR-catalyzed reduc- 17. Ikeda R, Nagao T, Okabe H, Nakano Y, Matsunaga H, Katano M, tion was similar to that of Forsythia PLR 3s and A. lappa Mori M (1998) Antiproliferative constituents in Umbelliferae ripening seed PLR. 26 plants. IV. Constituents in the fruits of Anthriscus sylvestris HOFFM. Chem Pharm Bull (Tokyo) 46:875-878 Lignan formation by the Anthriscus enzyme preparation 18. Ikeda R, Nagao T, Okabe H, Nakano Y, Matsunaga H, Katano M, along with the detection of lariciresinol and secoisolari- Mori M (1998) Antiproliferative constituents in Umbelliferae ciresinol from the plant suggests strongly that the conver- plants. III. Constituents in the root and the ground part of Anth- sion pinoresinol ~ lariciresinol --+ secoisolariciresinol is riscus sylvestris HOFFM. Chem Pharm Bull (Tokyo) 46:871-874 19. Koh D, Lim Y (1999) Anti-allergic activities of anthricin and its operating in A. sylvestris, as in Forsythia spp. 2'4~ Although structure elucidation. Agric Chem Biotechnol 42:208-209 Dewick et al. reported the in vivo conversion of matai- 20. Xia ZQ, Costa MA, Proctor J, Davin LB, Lewis NG (2000) resinol to podophyllotoxin via yatein and deoxypodophy- Dirigent-mediated podophyllotoxin biosynthesis in Linum flavum llotoxin,23'24'41 a detailed pathway after secoisolariciresinol to and Podophyllum pe#atum. Phytochemistry 55:537-549 21. Weiss SG, Tin-Wa M, Perdue RE Jr, Farnsworth NR (1975) Poten- yatein via matairesinol awaits enzymatic experiments in A. tial anticancer agents. II. Antitumor and cytotoxic lignans from sylvestris, which are underway in our laboratory. Linum album (Linaceae). J Pharm Sci 64:95-98 22. Lim YH, Leem MJ, Shin DH, Chang HB, Hong SW, Moon EY, Acknowledgments This research was partly supported by Grants-in- Lee DK, Yoon SJ, Woo WS (1999) Cytotoxic constituents from the Aid for Scientific Research (10660163, 12660150) and the Encourage- roots of Anthriscus Sylvestris. Arch Pharm Res 22:208-212 ment of Young Scientists (4542) from the Ministry of Education, 23. Jackson DE, Dewick PM (1984) Biosynthesis of Podophyllum Culture, Sports, Science and Technology of Japan; and by the lignans. II. Interconversions of aryltetralin lignans in Podophyllum hexandrum. Phytochemistry 23:1037-1042 Snmitomo Foundation. The authors are grateful to Dr. Guy Gusman, 24. Kamil WM, Dewick PM (1986) Biosynthetic relationship of Universit6 Libre de Bruxelles, Belgium, for his gift of Anthriscus aryltetralinlactone lignans to dibenzylbutyrolactone lignans. sylvestris seeds, and to Dr. Shingo Kawai, Faculty of Agriculture, Gifu Phytochemistry 25:2093-2102 University, Japan, for the (+_)-4-demethylyateinbenzyl ethers. 25. Erdtman H, Harmatha J (1979) Phenolic and terpenoid heartwood constituents of Libocedrus yateensis. Phytochemistry 18:1495-1500 26. Suzuki S, Umezawa T, Shimada M (1998) Stereochemical differ- ence in secoisolariciresinol formation between cell-free extracts References from petioles and from ripening seeds of Arctium lappa L. Biosci Biotechnol Biochem 62:1468-1470 27. Umezawa T, Kuroda H, Isohata T, Higuchi T, Shimada M (1994) 1. Umezawa T, Okunishi T, Shimada M (1997) Stereochemical diver- Enantioselective lignan synthesis by cell-free extracts of Forsythia sity in lignan biosynthesis. Wood Res 84:62-75 koreana. Biosci Biotechnol Biochem 58:230-234 541

28. Okunishi T, Umezawa T, Shimada M (2000) Enantiomeric compo- reductases from Forsythia intermedia. J Biol Chem 268:27026- sitions and biosynthesis of Wikstroemia sikokiana lignans. J Wood 27033 Sci 46:234-242 36. Katayama T, Davin LB, Chu A, Lewis NG (1993) Novel benzylic 29. Umezawa T, Shimada M (1994) Syntheses of (_+)-lariciresinols. ether reductions in lignan biogenesis in Forsythia intez~edia. Mokuzai Gakkaishi 40:231-235 Phytochemistry 33:581-591 30. Umezawa T, Isohata T, Kuroda H, Higuchi T, Shimada M (1992) 37. Umezawa T, Davin LB, Lewis NG (1990) Formation of the Biosynthesis of lignans. In: Kuwahara M, Shimada M (eds) Bio- lignan, (-)-secoisolariciresinol, by cell free extracts of Forsy- technology in pulp and paper industry. Uni Publishing, Tokyo, thia intermedia. Biochem Biophys Res Commun 171:1008- pp 507-512 1014 31. Okunishi T, Umezawa T, Shimada M (2001) Isolation and enzy- 38. Umezawa T, Davin LB, Lewis NG (1991) Formation of lignans matic formation of lignans of Daphne genkwa and Daphne odora. (-)-secoisolariciresinol and (-)-matairesinot with Forsythia J Wood Sci 47:383-388 intermedia cell-free extracts. J Biol Chem 266:10210-10217 32. Bradford M (1976) A rapid and sensitive method for the 39. Katayama T, Davin LB, Lewis NG (1992) An extraordinary accu- quantitation of microgram quantities of protein utilizing the prin- mulation of (-)-pinoresinol in cell-free extracts of Forsythia inter- ciple of protein-dye binding. Anal Biochem 72:248-254 media: evidence for enantiospecific reduction of (+)-pinoresinot. 33. Pelter A (1968) The mass spectra of some lignans of the 1-phenyl- Phytochemistry 31:3875-3881 1,2,3,4,-tetrahydronaphthatene series. J Chem Soc (c):74-79 40. Lewis NG, Davin LB (1999) Lignans: biosynthesis and function. In: 34. Umezawa T, Shimada M (1996) Formation of the lignan (+)- Sankawa U (ed) Comprehensive natural products chemistry, vol 1. secoisolariciresinol by cell-free extracts of Arctium lappa. Biosci Elsevier Science, Oxford, pp 639-712 Biotechnol Biochem 60:736-737 41. Broomhead AJ, Rahman MMA, Dewick PM, Jackson DE, Lucas 35. Chu A, Dinkova A, Davin LB, Bedgar DL, Lewis NG (1993) JA (1991) Matairesinol as precursor of Podophyllum lignans. Stereospecificity of (+)-pinoresinol and (+)-lariciresinol Phytochemistry 30:1489-1492